Use of cul4b activators in the preparation of anti-influenza virus drugs

Abstract

The application provides application of a CUL4B activator in preparation of an anti-influenza virus drug and belongs to the technical field of medicines. The application provides application of the CUL4B activator in preparation of the anti-influenza virus drug, wherein the action target point of the CUL4B activator comprises an E3 ligase complex formed by a CUL4B-DDB1-RBX1 complex and a DCAF (DDB1 and CUL4 associated factor) family protein. The application provides a new drug and a path for effective treatment of influenza.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and particularly relates to application of CUL4B activator in preparation of anti-influenza virus drugs. BACKGROUND

[0002] Influenza virus belongs to Orthomyxoviridae, and is a kind of enveloped negative-sense single-stranded RNA virus. Influenza virus can cause infection in hosts including humans, birds, pigs and horses. In the classification of virology, influenza virus is divided into four types A, B, C and D (A, B, C and D). According to the different antigenicities of viral surface hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA), the influenza virus of type A can be further divided into several subtypes, such as H1N1, H3N2, H5N1, etc. The influenza virus of type A mainly with H1N1 and H3N2 and the influenza virus of type B cause seasonal influenza. Influenza virus repeatedly causes human respiratory diseases every year, which has caused heavy burden to social economy and human health. According to the data published by WHO, the incidence of influenza in adults is 5%-10%, and the incidence in children is 20%-30%, and the number of deaths related to seasonal influenza reaches 650,000 per year. The symptoms of influenza mainly include cough, fever, chills, headache, etc.

[0003] The most effective way to prevent influenza is to inoculate corresponding vaccine. Although the vaccine has been used to prevent severe diseases and deaths caused by influenza virus, there are still some obstacles in practice. At present, the main influenza vaccines approved for marketing are mainly inactivated vaccine and attenuated live vaccine. Due to the rapid antigenic variation of influenza virus, multiple subtypes and the difference of human individual immune response, the protective effect of vaccine in specific population is not satisfactory. The vaccine only has preventive effect on existing virus subtypes, and has no effect on new strains generated by antigenic drift or antigenic shift. It is difficult to keep the vaccine consistent with the strain that will be prevalent. Therefore, effective antiviral small molecule drugs become a good choice for intervention of influenza virus infection. At present, four kinds of antiviral drugs have been approved for influenza treatment, including neuraminidase (NA) inhibitors (oseltamivir, zanamivir and peramivir) and PA inhibitors (baloxavir). However, due to the emergence of drug-resistant strains, antiviral drugs gradually lose their efficacy. For example, oseltamivir-resistant H1N1 strains have appeared in the 2007-2008 influenza season. Therefore, it is urgent to find new influenza virus treatment drugs. SUMMARY

[0004] In view of the above deficiencies of the prior art, the application provides application of a CUL4B activator in preparation of an anti-influenza virus drug.

[0005] To achieve the above object, the specific technical solutions of the application are as follows:

[0006] In a first aspect, the application provides application of a CUL4B activator in preparation of an anti-influenza virus drug.

[0007] The amino acid sequence of the CUL4B is shown in SEQ ID NO. 1.

[0008] Further, the action target of the CUL4B activator includes an E3 ligase complex formed by a CUL4B-DDB1-RBX1 complex and a DCAF (DDB1 and CUL4 associated factor) family protein.

[0009] Further, the CUL4B activator includes Etoposide (Etoposide) or a pharmaceutically acceptable salt thereof.

[0010] The structural formula of the Etoposide is as follows:

[0011]

[0012] In a second aspect, the application provides an anti-influenza virus drug, wherein the effective component of the drug is a CUL4B activator.

[0013] Further, the action target of the drug includes an E3 ligase complex formed by a CUL4B-DDB1-RBX1 complex and a DCAF family protein.

[0014] Further, the CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

[0015] Further, in a mouse experiment, the effective amount of the Etoposide is 10-50 mg / kg per day.

[0016] Further, the drug further includes a pharmaceutically acceptable carrier and / or excipient.

[0017] Further, the dosage form of the drug includes, but is not limited to, one of a tablet, a granule, a capsule, a dripping pill, a sustained-release agent, an oral liquid preparation, and an injection.

[0018] Compared with the prior art, the application has the following advantages:

[0019] 1. The application provides application of a CUL4B activator in preparation of an anti-influenza virus drug, wherein the action target of the CUL4B activator comprises an E3 ligase complex formed by a CUL4B-DDB1-RBX1 complex and a DCAF (DDB1 and CUL4 associated factor) family protein, silencing of any one of the proteins CUL4B, DDB1 and RBX1 can promote replication of an influenza virus PA protein, and the CUL4B activator can promote degradation of the PA protein, and the application provides a new drug and a path for effective treatment of influenza.

[0020] 2. The application is verified by cell level experiment research that the CUL4B activator Etoposide can enhance K48 type polyubiquitination of an influenza virus polymerase PA subunit, promote ubiquitin-proteasome degradation of PA, reduce virus polymerase activity, and thus inhibit infection of the influenza virus; the in-vivo experiment verifies that the CUL4B activator Etoposide has small toxicity and shows very good antiviral activity on the influenza virus, can significantly inhibit replication of the influenza virus in lung tissues of mice, and reduce lung injury caused by the virus, and has a wide application prospect in prevention and treatment of influenza virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For degradation of the influenza virus polymerase PA by the CUL4B-DDB1-RBX1 complex, the DCAF family protein serves as a substrate recognition acceptor and forms a CUL4B DCAF E3 ubiquitin ligase targeting PA degradation experiment results; wherein, Figure 1 A is the influence of knocking down CUL4A, CUL4B, DDB1 and RBX1 on the PA protein level, respectively; Figure 1 B is the influence of knocking down CUL4A, CUL4B, DDB1 and RBX1 on the PB2 protein level, respectively; Figure 1 C is the influence of knocking down CUL4A, CUL4B, DDB1 and RBX1 on the PB1 protein level, respectively; Figure 1 D is the DCAF family protein serving as a substrate recognition acceptor and forming a CUL4B DCAF E3 ubiquitin ligase targeting PA degradation model;

[0022] Figure 2 Etoposide in-vitro anti-influenza virus activity experiment results;

[0023] Figure 3 Etoposide anti-influenza virus mechanism research results; wherein, Figure 3A is the result of Western blot analysis of HEK293T cells after treatment with proteasome inhibitor MG132 or Etoposide; Figure 3 B is the result of Western blot analysis of HEK293T after treatment with Etoposide; Figure 3 C is the result of dual luciferase activity of HEK293T cells after treatment with Etoposide;

[0024] Figure 4 is the result of Etoposide in vivo anti-influenza virus activity experiment; wherein, Figure 4 A is an influenza virus infected mouse model; Figure 4 B is the body weight change of mice within 6 days of oral administration of Etoposide; Figure 4 C is the body weight change of mice within 6 days of influenza virus infection; Figure 4 D is the result of RT-qPCR experiment of lung tissue of mice; Figure 4 E is the result of HE staining of lung tissue of mice on the 6th day of infection. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] The present application provides the use of a CUL4B activator in the preparation of an anti-influenza virus drug.

[0027] The amino acid sequence of CUL4B is shown in SEQ ID NO. 1.

[0028] The target of the CUL4B activator includes the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

[0029] The CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

[0030] In the following specific embodiments, the structural formula of Etoposide is as follows:

[0031]

[0032] Example 1 Influence of CUL4B on influenza virus

[0033] 1. Influence of CUL4B-DDB1-RBX1 complex on influenza virus PA protein

[0034] Cullin4-RING (CRL4) E3 ligase is a multi-protein complex that can specifically recognize target proteins and ubiquitinate-dependent degradation. CRL4 E3 ubiquitin ligase is composed of Cullin4 (CUL4A / CUL4B), DNA damage-binding protein-1 (DDB1) and RING-box protein 1 (RBX1). CUL4A or CUL4B acts as a scaffold protein, recruiting DDB1 and RBX1 to assemble the DDB1-CUL4-RBX1 complex. Among them, RBX1 as a RING finger protein binds to E2 ubiquitin conjugase, while DDB1 as an adapter recruits DDB1 and CUL4-associated factors (DDB1- and CUL4-associated factors, DCAFs) to form a complete Cullin4-RING E3 ubiquitin ligase complex. DCAFs are responsible for recognizing substrates as substrate recognition receptors in Cullin4-RING E3 ubiquitin ligase and determining substrate specificity. This embodiment uses siRNAs to identify whether CUL4A or CUL4B plays a role in the degradation of influenza virus PA protein.

[0035] In HEK293T cells, siRNAs targeting host CUL4A, CUL4B, DDB1 and RBX1 or negative control (NC) and Flag-tag labeled PA expression plasmid were transfected respectively, and Western blotting was performed 48 h after expression. The experimental results are shown in Figure 1 A. Figure 1 A shows that the knockdown efficiency of siRNAs is about 70%-90%, compared with the control group, after knocking down CUL4B, the expression level of PA protein is significantly increased, while knocking down CUL4A has no significant effect on the expression level of PA protein, which indicates that CUL4B is responsible for regulating the degradation of PA protein in influenza virus. In addition, knocking down DDB1 or RBX1 also significantly increases the expression level of PA protein. The above results show that the CUL4B-DDB1-RBX1 complex is involved in the regulation of the stability of influenza virus PA protein expression.

[0036] 2, Effect of CUL4B-DDB1-RBX1 complex on influenza virus PB1 or PB2 protein

[0037] In HEK293T cells, siRNAs targeting host CUL4A, CUL4B, DDB1 and RBX1 or negative control (NC) and Flag-tag labeled PB1 or PB2 protein expression plasmid were transfected respectively, and Western blotting was performed 48 h after expression. The experimental results are shown in Figure 1 B andFigure 1 C. Figure 1 B and Figure 1 C shows that knockdown of DDB1-CUL4-RBX1 complex has no significant effect on the expression level of PB1 / PB2 protein compared with the control group, indicating that the CUL4B-DDB1-RBX1 complex has no obvious effect on the expression stability of PB1 or PB2.

[0038] 3, In addition to the CUL4B-DDB1-RBX1 complex, the formation of the complete CUL4B-RING E3 ubiquitin ligase also requires substrate recognition receptors DCAFs. And DDB1 can act as an adaptor to recruit DDB1 and CUL4-associated factors (DDB1- and CUL4-associated factors, DCAFs). In summary, these results show that the host DCAF family proteins, CUL4B, DDB1 and RBX1 form a complete CRL4B DCAF E3 ubiquitin ligase complex responsible for the degradation of polymerase PA protein (as shown in Figure 1 D).

[0039] As can be seen from the above, the CUL4B-DDB1-RBX1 complex is involved in the degradation of influenza virus polymerase PA, and the DCAF family protein acts as a substrate recognition receptor to form a CUL4B DCAF E3 ubiquitin ligase targeting PA degradation with CUL4B-DDB1-RBX1 complex.

[0040] Example 2 Etoposide in vitro anti-influenza virus activity and mechanism of action

[0041] 1, Etoposide in vitro anti-influenza virus activity

[0042] The anti-viral activity of Etoposide was evaluated by the reduction of intracellular influenza virus nucleoprotein (NP) after A549 cell infection. A549 cells were seeded in 12-well plates, and when the cell density reached about 90%, they were infected with influenza H1N1 PR8 virus (MOI=0.1) at 37℃ for 1 h, then the virus-containing medium was removed and the medium containing Etoposide (50 μM) was added; after 24 h of culture, the cells were lysed to obtain protein samples, and the virus NP and PA protein levels were detected by Western blotting using NP and PA specific antibodies to reflect virus replication and confirm the effect of Etoposide on virus infection.

[0043] The results of the experiment of Etoposide in vitro anti-influenza virus activity are shown in Figure 2 . As can be seen from the above, Figure 2 Etoposide has good anti-influenza virus activity at the cellular level.

[0044] 2. Mechanism of Etoposide against influenza virus

[0045] The mechanism of Etoposide in inhibiting influenza virus infection was explored by cell and biochemical experiments. Flag-PA expression plasmid (1 μg) was transfected into HEK293T cells, and 36 h after expression, the cells were treated with proteasome inhibitor MG132 (20 μM) or Etoposide (50 μM) for 12 h, followed by Western blot analysis. The results are shown in Figure 3 A. Autophagy-lysosome pathway and ubiquitin-proteasome pathway are the main protein degradation pathways in eukaryotic cells, and MG132 is a proteasome inhibitor. As can be seen from Figure 3 A, the use of MG132 alone can up-regulate the expression of influenza virus polymerase PA protein; the use of Etoposide alone can promote the degradation of PA protein; when MG132 and Etoposide are used simultaneously, MG132 blocks the degradation-promoting effect of Etoposide. This result shows that Etoposide can degrade PA through the ubiquitin-proteasome pathway, reducing its protein level.

[0046] Subsequently, it was detected whether Etoposide promotes the degradation of viral PA subunit by increasing the ubiquitin binding of the subunit. Flag-PA and HA-Ub (K48) expression plasmids or empty vector were co-transfected into HEK293T cells for expression for 36 h, and then treated with Etoposide (50 μM) for 12 h. After the cells were lysed with RIPA lysis buffer, immunoprecipitation (IP) experiment was performed with Flag antibody, and the immunoprecipitate was used for Western blot analysis. The results are shown in Figure 3 B, which shows that Etoposide increases the K48 type ubiquitination of PA protein.

[0047] The effect of Etoposide on the overall replication and transcription of the virus was detected using an influenza virus minireplicon system. The minireplicon system plasmid was co-transfected into HEK293T cells, and 36 h after transfection, the cells were treated with Etoposide (50 μM) for 12 h (DMSO as control), and the dual luciferase activity of the cells was detected. The results are shown in Figure 3 C, which shows that Etoposide significantly reduces the activity of influenza virus polymerase.

[0048] The above results show that Etoposide can enhance the K48 type polyubiquitination of influenza virus polymerase PA subunit, promote the ubiquitin-proteasome degradation of PA, reduce the activity of viral polymerase, and thus inhibit the infection of influenza virus.

[0049] Example 3 Etoposide in vivo anti-influenza virus activity

[0050] Etoposide down-regulates the expression of influenza virus PA protein in vitro, limiting the infection of influenza virus, while the effect in vivo in mice has not been identified. Next, the anti-influenza virus activity of Etoposide in vivo was verified.

[0051] The mouse model of influenza virus infection is as shown in Figure 4 A, Etoposide (20 mg / kg) was orally administered to 6-8 week old C57BL / 6J mice for 6 consecutive days, once a day, and the mice were infected with mouse-adapted PR8 virus through the nasal route after being anesthetized with isoflurane anesthetic. The body weight of the mice was monitored for several consecutive days, and the mice were euthanized on the 6th day of infection, and the lung tissues were taken for virus load determination and pathological observation. The mice were only orally administered with drugs, and the body weight of the mice was monitored for several consecutive days to determine the in vivo toxic effect of Etoposide. The experimental results are shown in Figure 4 B, 4C; wherein, Figure 4 B shows that no significant decrease in body weight was observed in mice receiving Etoposide (20 mg / kg) for 6 consecutive days, indicating that Etoposide has no significant drug toxicity in mice in vivo; Figure 4 C shows that mice not receiving Etoposide oral administration exhibited more significant body weight loss after virus infection than mice receiving Etoposide oral administration.

[0052] In order to further understand the reason why Etoposide can reduce the body weight loss of infected mice, the lung tissues of mice on the 6th day of infection were taken, crushed by a tissue crusher, and then the RNA was extracted by TRIzol reagent, and then the lung virus load was detected by RT-qPCR. The experimental results are shown in Figure 4 D, the figure shows that the abundance of virus NP RNA in the lung tissues of mice in the Etoposide treatment group was significantly lower than that in the control group, indicating that Etoposide treatment can reduce the virus load in the lung tissues of mice. At the same time, the lung tissues of mice on the 6th day of infection were subjected to HE staining. The experimental results are shown in Figure 4 E, the figure shows that compared with the control group, the lung tissue of mice not receiving Etoposide oral administration has more severe pathological changes, more cell and protein debris infiltration in the alveoli, and obvious alveolar necrosis.

[0053] The above results demonstrate that Etoposide can inhibit the replication of influenza virus in mice in vivo and reduce the lung damage caused by influenza virus.

[0054] The above detailed description describes the implementation of the present application, but the present application is not limited to the specific details in the above implementation. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. Application of a CUL4B activator in the preparation of an anti-influenza virus drug, characterized in that: The CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

2. Use of the CUL4B activator according to claim 1 in the preparation of anti-influenza virus drugs, characterized in that: The targets of the CUL4B activator include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

3. Use of the CUL4B activator according to claim 1 in the preparation of anti-influenza virus drugs, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

4. Use of the CUL4B activator according to claim 1 in the preparation of anti-influenza virus drugs, characterized in that: The dosage forms of the drug include tablets, granules, capsules, dripping pills, sustained-release preparations, oral liquid preparations or injections.

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